It is considered a demanding problem to define and develop a method for preparing a composition of a protein with pharmaceutical properties, suitable for large scale pharmaceutical production and resulting in a safe, efficient and clinically acceptable pharmaceutical product. One problem is to retain the stability of the protein during preparation, storage and handling. Another problem is to assure the desired pharmacokinetic and pharmacodynamic properties of the protein. It has frequently been suggested that dispersed lipid systems would constitute suitable carriers for pharmaceuticals including proteins and the mentioned problems are in many aspects the same also for such dispersed systems. It would therefore be desirable to be able to provide a method of linking proteins and lipids and thereby overcoming the mentioned problems which is many cases also relates to the dispersed lipids. In particular, it would be desirable to be able to associate the protein with a lipid carrier for improving the protein stability and the provision of a specifically designed drug delivery formulations. In certain applications, it would for example also be desirable to extend the in-vivo half life in the circulation system of the bioactive protein which otherwise risks to be enzymatically degraded before reaching the target where it should exert its beneficial activity.
A lot of efforts has been devoted in finding such suitable administration forms which maintain the bioactivity of the protein, while at the same time avoiding the involvement of carriers and formulation adjuvants which can cause clinical side-effects. Imitations of the native lipid-protein transport particles in the blood stream have been suggested as one attractive model for designing administration systems for bioactive proteins. Some important forms of these lipid particles are chylomicrons, the transporters of triglycerides which appear in the blood stream after ingestion of lipid rich food, VLDL, LDL- and HDL-particles. These particles are mainly composed of free and esterified cholesterol, triglycerides, phospholipids and several other minor lipid components and proteins. The LDL-particles serve as transporters of cholesterol and other lipids to the cells, while the HDL-particles transport these materials to the liver for elimination. A HDL particle frequently has a disc-shaped form with an outer surface covered by a phospholipid layer and a hydrophobic core. Amphiphilic proteins, such as apolipoprotein A-I and A-II are attached to the surface by means of interaction of the hydrophobic face of their alpha helical domain with the hydrophobic part of the phospholipids.
Synthetic chylomicron-like products have particularly found use as parenteral nutrients. It is a widely established technology to prepare lipid emulsions from a purified triglyceridic oil (predominantly soybean and safflower oil) and phospholipids (from egg yolk or soybeans) which are regarded as clinically acceptable for parenteral use due to their chylomicron-like emulsion droplets, generally of the size between 0.1 to 1 .mu.m. There also exist several commercial products where such emulsions are used as carriers for lipophilic drugs which are dissolved in the dispersed lipid phase, such as Diazemuls.RTM. and Diprivan.RTM.. However, a practical complication with this type of emulsion carriers is their relative physical instability which frequently is impaired by the addition of the hydrophobic drug and lead to a break-up of the emulsion and thus making it dangerous to administer because of the risk of lipid embolism. There have been many attempts to solve this problem by adding stabilizers which, however, often are connected with undesired side-effects. The liability of such emulsions, also in connection with sterilization by high pressure steam, i.e. autoclavation and during subsequent storage, have often inhibited their use as drug parenteral drug carriers. Generally, autoclavation procedures also tend to damage many labile pharmaceuticals to be incorporated with emulsions as for example many proteins.
Liposomes have frequently been suggested as suitable vehicles for parenteral protein delivery, as for example disclosed in the article by A L Weiner in Immunomethods, 1994, Vol. 4, pp. 201-209. A liposome carrier would for example be advantageous when an improved solubilization, a sustained release (or extended half-life) or an improved targeting of the protein are desired. It is, however, acknowledged in the mentioned article that many frequently methods used to design liposomal systems often involve procedures which risk to destroy the activity of sensitive proteins, for example by denaturation and oxidation. Moreover, in Liposome Technology, 1993 by CRC Press Inc., Vol. 1, Ch. 3, pp. 49-63: M M Brandl et al, it is disclosed how to utilize high pressure homogenization for preparing liposomes of small unilamellar quality and suitability of this technique for the reduction of vesicle size, broadness of size distribution and lamellarity of preformed multilamellar vesicle dispersions. Also the entrapment of proteins and peptides, specifically hemoglobin and insulin, is disclosed, however, the small size of the resulting vesicles is disadvantageous and the entrapment efficiencies of the proteins are low. Moreover, it is reported that the integrity and the biological function of hemoglobin is maintained, at least during short exposures to stressing conditions.
Another type of delivery system of a dispersed lipid agent which is suggested as suitable for proteins is disclosed in WO 93/06921. This system comprises colloidal lipid particles having an interior non-lamellar phase of lipids such as a reversed hexagonal phase or cubic phase which may be associated with a protein.
On the other hand, many proteins in purified form are notoriously difficult to formulate. For example, human growth hormone (hGH) exhibits poor stability in aqueous solution during storage for which reason it is advised to store preparations in a lyophilized form until its administration when it is reconstituted to an injectible solution. However, an inadvertent exposure to shear forces due to a careless reconstitution process will irrevocably lead to a loss of biological activity. For this reason especially designed means for performing a gentle reconstitution have been developed for human growth hormone as disclosed in EP 0 298 067.
There are many disclosures of synthetic HDL-particles in the literature which refer to their capacity in picking up and removing undesired lipid material in the blood stream and from the blood vessels thus making them potentially useful in therapy for treating atherosclerosis by depleting cholesterol from arterial plaques and for removing lipid soluble toxins such as endotoxins.
In Experimental Lung Res. 1984, Vol. 6, pp. 255-270: A Jonas, experimental conditions of forming complexes of the partially hydrophobic apolipoproteins and phospholipids are described in detail. It was found that, by contacting apolipoproteins with preformed phosphatidyl choline vesicles, lipid particles were spontaneously formed which could be used as analogs of HDL-particles. By mixing phosphatidyl choline and bile acids to a miscellar dispersion and contacting the resultant mixture with apolipoproteins specifically shaped, discoidal and thermodynamically stable lipid particles were formed by means of a dialysis method, subsequently called the "cholate-dialysis method".
U.S. Pat. No. 4,643,988 to Research Corporation describes synthetic peptides useful in treatment of atherosclerosis with an improved amphiphatic helix and an ability to spontaneously form stable discoidal lipid particles with phospholipids which resemble native HDL-complexes. The lipid particles can be formed by contacting vesicles of phosphatidyl choline made by sonication. However, such a production method including sonication is suitable only for smaller batches of lipid particles and not for large scale pharmaceutical production.
U.S. Pat. No. 5,128,318 to Rogosin Institute describes the production of reconstituted lipoprotein containing particles (HDL-particles) from plasma derived apolipoproteins which are processed to synthetic particles for parenteral administration with the addition of cholate and egg yolk phosphatidyl choline. A similar method is also disclosed in the Japanese patent application JP 61-152632 to Daiichi Seiyaku KK.
Also in WO 87/02062 to Biotechn. Res. Partners LTD, it is disclosed how to obtain a stabilized formulation by incubating a solution of recombinantly produced lipid binding protein, such as human apolipoprotein, with a conventional lipid emulsion for parenteral nutrition.
The article by G. Franceschini et al. in J. Biol. Chem., 1985, Vol. 260 (30), pp. 16231-25 considers the spontaneous formation of lipid particles between apolipoprotein A-I and phosphatidyl choline. In this article, it is also revealed that Apo-IM (Milano), the variant of apolipoprotein A-I carried by individuals shown to have a very low prevalence of atherosclerosis, has a higher affinity (association rate) to dimyristoyl phosphatidyl choline (DMPC) than regular Apo A-I. It is suggested that the mutant Apo A-IM has a slightly higher exposure of hydrophobic residues which may contribute both an accelerated catabolism and an improved tissue lipid uptake capacity of such Apo A-IM/DMPC particles.
The Canadian patent application CA 2138925 to the Swiss Red Cross discloses an improved, more industrially applicable, method of producing synthetic reconstituted high density lipoprotein (rHDL) particles from purified serum apolipoproteins and phospholipids which avoids organic solvents while resulting in less unbound, free non-complexed phospholipids (i.e. a higher yield of lipoprotein particles). Herein, it is suggested to mix an aqueous solution of apolipoproteins with an aqueous solution of phospholipid and bile acids, whereupon the resultant mixture is incubated and protein-phospholipid particles are spontaneously formed when bile acids are removed from phospholipid/bile acid micelles with diafiltration.
The method employing the use of bile acids for making a micellar dispersion of the lipid according to the cholate-dialysis method have several drawbacks for lipid particle production, since it requires a specific separation step from the resultant mixture. Additionally bile acid residues may even in small amounts be suspected to induce side-effects after parenteral administration and may also constitute a risk of viral contamination. Moreover, the methods referred to above for preparing lipoprotein-lipid particles generally suffer from poor reproducibility and non-definable particle sizes. In particular, none of these methods are suitable in large-scale industrial process under well-controlled conditions.
A surprisingly advantageous method is demonstrated by the present invention which meets these requirements and solve numerous problems which otherwise are associated with protein formulation, especially in large-scale production.